The terpenoids constitute the largest family of natural products, and play diverse functional roles in plants as hormones, photosynthetic pigments, electron carriers, mediators of polysaccharide assembly, and structural components of membranes. In addition, many specific terpenoid compounds serve in communication and defense. Some terpenoids, available in relatively large amounts, are important renewable resources and provide a range of commercially useful products. Members of the terpenoid group also include industrially useful polymers and a number of pharmaceuticals and agrochemicals.
The biosynthesis of terpenoids can be divided into four major processes, the first of which involves the conversion of acetyl-coenzyme A (CoA) to the “active isoprene unit”, isopentenyl pyroposphate (IPP). By the action of various prenyltransferases this precursor is transformed into higher order terpenoid building blocks, geranyl pyrophosphate (GPP, C10), farnesyl pyrophosphate (FPP, C15), and geranylgeranyl pyrophosphate (GGPP, C20). These branch point intermediates may then self-condense (to the C30 and C40 precursors of sterols and carotenoids, respectively), be utilized in alkylation reactions to provide prenyl side chains of a range of non-terpenoids, or undergo internal addition to create the basic parental skeletons of the various terpenoid families (McGarvery and Croteau (1995) Plant Cell 7:1015-1026).
The initial steps of the pathway involve the fusion of three molecules of acetyl-CoA to produce the C6 compound 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The first two reactions are catalyzed by two separate enzymes, acetoacetyl-CoA hiolase and HMG-CoA synthase. Neither of these enzymes has been extensively studied in plants. The next step, catalyzed by HMG-CoA reductase, is of paramount importance in animals as the rate limiting reaction in cholesterol biosynthesis (for review, see Goldstein and Brown (1990) Nature 343:425-430). This enzyme catalyzes two reduction steps, each requiring NADPH. Reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis (Honda et al. (1998) J. Lipid Res. 39:44-50).
The mevalonate resulting from the reduction of HMG-CoA is sequentially phosphorylated by two separate kinases, mevalonate kinase, and phosphomevalonate kinase, to form 5-pyrophosphomevalonate. Formation of IPP is then catalyzed by pyrophosphomevalonate decarboxylase, which performs a concerted decarboxylative elimination. This enzyme requires ATP and a divalent metal ion. The tertiary hydroxyl group of pyrophosphomevalonate is phosphorylated before the concerted elimination, thus making a better leaving group (McGarvery and Croteau (1995) Plant Cell 7:1015-1026). IPP is the basic C5 building block that is added to prenyl pyrophosphate cosubstrates to form longer chains. IPP is first isomerized to the allylic ester dimehylallyl pyrophosphate (DMAPP) by IPP isomerase.
Isoprene is synthesized directly from DMAPP by diphosphate elimination in a reaction catalyzed by isoprene synthase. Higher terpenoids are generated by the action of prenyltransferases which perform multistep reactions beginning with DMAPP and IPP to form higher isoprenologs. GPP synthase forms the C10 intermediate (GPP) from DMAPP and IPP. This synthase has been characterized in a number of plant species (Croteau and Purkett (1989) Arch. Biochem. Biophys. 271:524-535). FPP synthase forms the C15 intermediate (FPP) in two discrete steps: first DMPP and IPP form GPP which remains bound to the enzyme; then another IPP is added to yield FPP (McGarvery and Croteau (1995) Plant Cell 7:1015-1026).
The enzymes forming the HMG-CoA leading to ketone bodies occur in the mitochondria whereas those responsible for the synthesis of the HMG-CoA that is destined for sterol biosynthesis are located in the cytosol. Their catalytic mechanisms, however, are identical. HMG-CoA reductase has been localized to plastids and mitochondria in radish (Bach (1986) Lipids 21:82-88; Bach (1987) Plant Physiol Biochem. 25:163-178) although the Arabidopsis enzyme is thought to be localized only to the endoplasmic reticulum (Enjuto et al. (1994) Proc. Natl. Acad. Sci. USA 91:927-931). Mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isopentenyl diphosphate isomerase, and farnesyl diphosphate (FPP) synthase are localized predominantly in peroxisomes (Lenka and Skaidrite (1996) J. Biol. Chem. 271:1784-1788).
Acetoacetyl-CoA C-acetyltransferase, also called acetoacetyl-CoA thiolase (EC 2.3.1.9), functions as a homotetramer, plays a major role in ketone body metabolism, and catalyzes the first step in the biosynthesis of poly beta-hydroxybutyrate. The gene encoding acetoacetyl-CoA thiolase has been identified in radish, and a sequence encoding a corn acetoacetyl-CoA thiolase is found in the NCBI database having General Identifier No. 5531937. The corn sequence corresponds to the C-terminal half of the radish sequence. EST sequences with similarities to those encoding acetoacetyl-CoA thiolases are found in the NCBI database having General Identifier Nos. 5607829, 6021192, 5607308, 3763023, 426049, 3107208, 2443029, and 5761368.
HMG-CoA synthase (EC 4.1.3.5) condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA. Cytosolic HMG-CoA synthase is a highly regulated enzyme involved in isoprenoid biosynthesis and therefore a potential target for cholesterol-lowering drugs (Russ et al. (1992) Biochim. Biophys. Acta 1132:329-331). The genes encoding HMG-CoA synthase have been identified in Arabidopsis thaliana and Pinus sylvestris but not in any crop species. EST sequences with similarities to those encoding HMG-CoA synthases are found in the NCBI database having General Identifier Nos. 5030550, 6012290, 5901402, 3760977, 2427448, 428081, and 454498.